Novel diethanolamine triacetic acid triamides and methods for improving feed utilization and lactation in ruminant animals

ABSTRACT

Novel diethanolaminetriacetic acid triamide compounds such as diethanolaminetriacetic acid tris(N,N-diisobutylamide) (alternatively named 2,2&#39;-[[bis(2-methylpropyl)amino]-2-oxoethyl]nitrilo]bis[2,1-ethanediyloxy]bis[N,N-bis (2-methylpropyl)acetamide]) are disclosed that are useful for improving feed utilization efficiency of ruminants and for improving lactation of lactating ruminants. Also disclosed are novel intermediates for preparation of the triamide compounds.

FIELD OF THE INVENTION

The invention relates to novel compounds and methods for improving feedutilization and lactation in ruminant animals. In particular, theinvention relates to nvel diethanolaminetriacetic acid triamides and tomethods of improving ruminant feed utilization and lactation byadministering one or more of said triamides.

BACKGROUND OF THE INVENTION

It is well established that improvements in feed utilization efficiencyin ruminant animals can be achieved by altering the fermentation processwhich takes place in the rumen. Ruminant animals utilize their food bydegrading the carbohydrates contained therein to pyruvate andmetabolizing the pyruvate to volatile fatty acids (VFAs) such asacetate, propionate and butyrate. These VFAs are absorbed from the gutand are employed for energy production which can than be channeled intogrowth, lactation, etc., by the ruminant.

The process of formation of acetate in the rumen is one of the majorinefficiencies in the digestive process. Since acetate is made by thedegradation of a pyruvate molecule, each molecule of acetate which isproduced is accompanied by a one carbon molecule which subsequentlyresults in the formation of methane. Most of the methane produced islost through eructation. Since butyrate is made from two molecules ofacetate, each molecule of butyrate involves the loss to the animal oftwo molecules of methane, with all of the associated energy.

Thus, the efficiency of carbohydrate utilization (carbohydrates beingthe major nutritive portion of ruminant animals' feed) can be increasedby treatments which encourage the animal to produce propionate ratherthan acetate or butyrate from carbohydrates. Further, the efficiency offeed use can be effectively monitored by observing the production andconcentration of propionate in the rumen. If the animal is making morepropionate, it will be found to be using its feed more efficiently. Areduction in the amount of methane produced has also been observed to bea good indication of increased feed efficiency.

In this regard, compounds which increase the amount of propionateproduced by a ruminant animal enhance the efficiency of feed utilizationand have been observed to provide many beneficial results. For instance,it has been disclosed that administration of certain antibiotics whichpromote production of propionate can be used to promote growth rates ofruminant animals (see, e.g., Celmer et al., U.S. Pat. No. 4,431,801;Maehr, U.S. Pat. No. 4,218,560). Additionally, propionate-increasingsubstances have also been helpful in improving milk production inlactating ruminants (e.g., Scheifinger, U.S. Pat. Nos. 4,430,328 and4,336,250). It is thus highly desirable to develop compounds which canincrease the production of propionate in ruminant animals such ascattle, sheep or goats in order to improve feed utilization by theanimals which will promote the growth of the animals and also achieveimproved lactation as well.

SUMMARY OF THE INVENTION

Novel diethanolaminetriacetic acid triamide compounds are provided whichare useful in improving the feed utilization efficiency in ruminantanimals such as cows, sheep or goats. The novel triamides also improvelactation in lactating ruminant animals. A method of increasing theefficiency of feed utilization is also provided which comprisesadministering to ruminant animals an effective amount of one or moretriamide compounds of the present invention. Also included is a methodfor improving lactation in lactating ruminants comprising administeringa lactation improving amount of one or more triamide compounds of thepresent invention. As used herein, the term "effective amount" refers tothat amount of one or more compounds of the invention, when administeredto a ruminant animal, that is sufficient to increse the growth rateand/or feed conversion efficiency of the treated ruminant animalswithout resulting in any significant adverse side effect; the term"lactation improving amount" refers to that amount of one or morecompounds of the invention, when administered to a lactating ruminant,that is sufficient to cause an observable improvement in milk productionwithout resulting in any significant adverse side effects.

DETAILED DESCRIPTION OF THE INVENTION

The triamide compounds of the present invention are of the followingformula: ##STR1## wherein each R may be the same or different andrepresents a straight or branched chain alkyl group having one to sixcarbon atoms. The physiologically acceptable salts of the triamidecompounds are also within the scope of the invention. Preferably, R willbe a group having three to five carbon atoms, such as tert-butyl,n-butyl, isopropyl or isobutyl. Most preferably, R will be isobutyl, andthe preferred compound of the present invention will bediethanolaminetriacetic acid tris (N,N-diisobutylamine).

The triamide compound of the present invention can be prepared by addingto a suspension of NaH in dimethoxyethane at about 0° C. a diolamidehaving the formula: ##STR2## wherein R is as disclosed above in FormulaI. To this suspension is added a chloroamide of the formula: ##STR3##wherein R is as described above. The reaction mixture is heated toreflux, a drop of water is added, and the solvent is removed. Theremaining organic residue layer is washed and concentrated to give thedesired triamide in the form of an oil.

The chloroamide (III) can be prepared by reaction of chloroacetylchloride and the appropriate amine in an inert solvent such as diethylether. This reaction is ilustrated as follows: ##STR4## wherein R is asdescribed above. The diolamide (compound of Formula II) is novel and istherefore also within the scope of the present invention.

The dichloroamide (II) can be prepared by heating a mixture ofdiethanolamine, the chloroamide (III) and a base such as triethylamine(Et₃ N) in an inert solvent such as dimethoxymethane (DME). Thisreaction is illustrated as follows: ##STR5## wherein R is as describedabove.

The salts of the compounds of this invention are physiologicallyacceptable salts derived from physiologically acceptable acids. Suchphysiologically acceptable acids include inorganic acids, e.g.,hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid,hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, andthe like, as well as organic acids, such as aliphatic mono- ordicarboxylic acids, phenyl-substituted alkanecarboxylic acids,hydroxyalkanecarboxylic acids or alkanedicarboxylic acids, aromaticacids, aliphatic or aromatic sulfonic acids, and the like.

Physioligically acceptable salts of these acids include therefore, forexample, the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide, fluoride,acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caproate, heptanoate, propionate, malonate, succinate,suberate, sebacate, fumarate, maleate, mandelate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,terephthalate, benzenesulfonate, toluenesulfonate,chlorobenzenesulfonate, xylenesulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate,beta-hydroxybutyrate, glycollate, malate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, andthe like.

The salts of the triamide of the present invention can be preparedaccording to procedures commonly employed for the preparation of anionicsalts. For example,t he free base form of a triamide compound isdissolved in a suitable solvent, and an aqueous or organic solution ofthe desired acid is added to the triamide solution. The triamide anionicsalts can be isolated by filtration and recrystallization or byevaporation of the solvent and purification.

In the present invention, an effective amount of triamide compound isadministered to a ruminant animal in order to improve the efficiency offeed utilization by the animal. It is well established that theadministration of substances which improve feed utilization efficiencythrough increases in propionate production also acts to improve growthrates in ruminant animals. The improved growth rates result from theadditional energy made available by the more efficient processing offeed. Therefore the feed utilization method of the invention alsoencompasses growth promotion activity.

It has been further established that propionate-increasing compoundswhich improve feed utilization efficiency can also be effective inincreasing milk production in lactating ruminant animals. In thelactating ruminant, energy for lactation is often the most limitingfactor in milk production, and improvements in feed utilizationefficiency often provide the energy needed for increased lactation.However, improving feed utilization by increasing the animal'sproduction of propionate will not always ensure that the milk producedby the lactating ruminant will be of sufficient quality. In milkproduction, acetate is required in order to synthesize milk fat, whilepropionate is utilized to produce glucose, and has very little effect onthe milk fat produced. In addition, butyrate is degraded into acetateunits and then it too can be used in long chain fatty acid synthesisproducing the milk fat.

Accordingly, in order to increase milk production in lactating ruminantsit is necessary to increase production of propionate, but not at theexpense of significant decreases in acetate or butyrate production.Significant reductions in acetate and butyrate production result in amilk product of reduced milk fat content, rendering the milk lesseconomically desirable in the United States (at the present time). It isnecessary, therefore, that a compound desirable for use in promotinglactation be one that improves propionate production withoutsignificantly lowering acetate and butyrate levels.

The triamide compounds of the present invention provide a significantincrease in propionate levels without causing a significant decrease inacetate or butyrate levels, and thus can be used to promote lactation inlactating ruminants. A method of improving lactation in ruminants isthereby provided which comprises administering to lactating ruminantanimals a lactation improving amount of one more triamide compounds ofthe present invention. The administration of the triamide(s) may bedirect, or a composition for promoting lactation may be prepared whichcomprises a lactation-promoting amount of the triamide of Formula I andan inert carrier.

It is not desired to be bound by any particular mechanism or theory;however, the triamide compounds of the present invention are believed tofunction by selectively inhibiting the growth of a number of species ofbacteria which are commonly found in the rumen of ruminant animals. Whenincluded in a growth medium designed to support the growth of pure rumenbacterial species, one or more triamides inhibit growth ofmicroorganisms such as Ruminococcus flavefaciens, Butyrivibriofibrisolvens, Streptococcus bovis, Bacteroides ruminicola and the like.In contrast, organisms such as Selenomonas ruminantium usually arepractically unaffected. In general, the triamide compounds of thepresent invention seem more active against gram-positive bacteria thangram-negative bacteria.

The triamide compounds of the present invention can be administered toincrease feed utilization efficiency of ruminants in any way whichensures that the animals receive an effective amount of one or more ofthe desired compounds. It is preferred that one or more compounds beadministered orally and at an effective amount of from about 0.2 toabout 100 milligrams (mg) per kilogram (kg) of ruminant body weight perday. It is particularly preferred that the effective amount is betweenabout 1 to about 10 mg/kg of body weight per day of one or more triamidecompounds of the present invention.

The triamide compounds of the present invention can be administered toimprove lactation of lactating ruminants in any way which ensures thatthe animals receive a lactation improving amount of one or more of thedesired compounds. A lactation improving amount is typically that amountthat results in an increase in the volume of milk produced by about 2 toabout 15 percent relative to untreated animals. It is preferred that oneor more compounds be administered orally and at a location improvingamount of from about 0.2 to about 100 mg per kg of ruminant body weightper day. It is particularly preferred that the lactation improvingamount is between about 1 and about 10 mg/kg of body weight per day ofone or more triamide compounds of the invention.

The exact effective amount and/or lactation improving amount of one ormore triamides to be employed will vary depending upon factors such asspecies of animal, or the size, weight, age and health of the animal. Inparticular cases, the concentration can be determined by conventionaldose titration techniques.

The most practical way to administer the triamide compound of thepresent invention is to add an effective amount (or lactation improvingamount) of one or more triamides or physiologically acceptable saltsthereof into the animal's feed. This can be done by directly adding adesired amount of the triamide(s) to the feed supply, or by firstforming the compound(s) into a premix composition for subsequentaddition to the feed. Such a composition preferably comprises one ormore triamide compounds of Formula I above, along with an inert carrier,the amount of triamide(s) being sufficient to result in increased feedutilization and/or improved lactation in an appropriate ruminant animalupon administration to said animal. Suitable carriers include groundcorn, barley, soybean meal, wheat, soy flour, or any similar low-priced,edible material. It is desired that the amount of one or more triamidecompounds used in the composition be sufficient to comprise from about 5to about 1000 parts per million in the animal feed. It is particularlypreferred that the triamide comprises from about 30 to about 300 partsper million of the ultimate animal feed.

The form of the additive to the feed is not crucial, and alternativeforms for administering the triamide compound may be employed. Forinstance, the compound can be incorporated into tablets, drenches, saltblocks, paste, boluses, or capsules and doped to the animals.Formulation of the compounds in such dosage forms can be accomplished bymeans of methods well known in the veterinary pharmaceutical art. Eachindividual dosage unit should contain a quantity of the compound whichhas a direct relation to the proper daily dose for the animal to betreated, as discussed above.

The following examples are provided in order to further illustrate thepresent invention and should not be construed as limiting the inventionin any way.

EXAMPLE 1 Preparation of Chloroacetic Acid, N,N-diisobutylamide

To a suspension of diisobutylamine (110 g, 149 mL), sodium carbonate (80g) and ether (400 mL) at -10° C. was added chloroacetyl chloride (85 g,0.75 mol). The reaction mixture was maintained below room temperatureduring the addition. The mixture was stirred for 45 minutes after theaddition was complete. The solid was removed by filtration, and thefiltrate was concentrated resulting in a residue. The residue wasdissolved in methylene chloride, extracted with 1N HCl followed bydilute sodium carbonate and dried over solid sodium sulfate whichresulted in an aqueous and organic layer. Concentration of the organiclayer afforded 80.8 g of a light yellow liquid. The NMR spectrum of thislight yellow liquid was consistent with the desired product andindicated that the compound was >95% pure.

EXAMPLE 2 Preparation of Diethanolamineacetic Acid, N,N-diisobutylamide

A mixture of diethanolamine (1.15 g, 0.011 mol), chloroamide 3 (2.0 g,0.01 mol), triethylamine (0.011 mol) and dimethoxyethane (20 mL) washeated under reflux for 24 hours. The solvent was removed, and theresidue was partitioned between 1N HCl and methylene chloride. Theaqueous layer was basified and extracted with ethyl acetate (heptanewould not extract the product). The organic layer was concentrated togive 2.0 g of a colorless oil. The FDMS, NMR spectrum, and the infraredspectrum of this oil were consistent with the desired product.

EXAMPLE 3 Preparation of Diethanolaminetriacetic AcidTris(N,N-diisobutylamide)

To a suspension of NaH (0.004 mol., 0.16 g) in dimethoxyethane (10 mL)at 0° C. was added a diolamide (0.55 g, 0.002 mol) having the formula:##STR6## wherein iBu represents isobutyl. To this mixture at roomtemperature was added a chloroamide (0.0042 mol, 0.86 g) having theformula: ##STR7## wherein iBu is isobutyl. The reaction mixture was thenheated to reflux for 45 minutes. A drop of water was added, and thesolvent was removed. The residue was partitioned between 1N HCl andmethylene chloride. The organic layer was washed with aqueous sodiumcarbonate and concentrated to afford 1.3 g of an oil. The resultingproduct was diethanolaminetriacetic acid tris(N,N-diisobutylamide)having the formula: ##STR8##

This compound can be alternatively named2,2'-[[2-[bis(2-methylpropyl)amino]-2-oxoethyl]nitrilo]bis[2,1-ethanediyloxy]bis[N,N-bis(2-methylpropyl)acetamide]. The structure was confirmed by fielddesorption mass spectrometry (FDMS), NMR spectroscopy, and infraredspectroscopy of the product.

EXAMPLE 4 Effect of Triamide Compound on Rumen Bacterial Growth

The triamide compound of Example 3 above was placed in a growth culturemedium of five rumen bacterial species, and growth of the treatedcultures was monitored and compared with growth in control media. Theresults are presented in Table 1 below. The triamide compound of thepresent invention prevented the growth of R. flavefaciens and B.fibrisolvens. In addition, the growth of S. bovis and B. ruminicola wasinhibited. The growth of S. ruminantium was almost unaffected by thetriamide. In general, the triamide appeared to be more active againstgram-positive bacteria than gram-negative bacteria.

                  TABLE 1                                                         ______________________________________                                        RESPONSES OF BACTERIA TO                                                      TREATMENT WITH TRIAMIDE                                                       BACTERIA        OD.sub.660.sup.1                                                                        SD.sup.2                                                                             RESPONSE.sup.3                               ______________________________________                                        Ruminococcus                                                                            control   1.131     0.033                                                                              0                                          flavefaciens                                                                            w/triamide                                                                              -0.008    0.008                                                                              101                                        Butyrivibrio                                                                            control   1.813     0.073                                                                              0                                          fibrisolvens                                                                            w/triamide                                                                              -0.033    0.009                                                                              102                                        Streptococcus                                                                           control   1.694     0.051                                                                              0                                          bovis     w/triamide                                                                              0.168     0.079                                                                              90                                         Bacteroides                                                                             control   1.648     0.011                                                                              0                                          ruminicola                                                                              w/triamide                                                                              0.116     0.071                                                                              93                                         Selenomonas                                                                             control   1.704     0.049                                                                              0                                          ruminantium                                                                             w/triamide                                                                              1.343     0.092                                                                              21                                         ______________________________________                                         .sup.1 Each value is the mean of triplicate samples.                          .sup.2 SD is the standard deviation.                                          .sup.3 Responses represent inhibition of growth as a percent of the           controls and are calculated as follows: Response = 100 ×                (1(experimental optical density at 660 nanometers (OD.sub.660)/control        OD.sub.660))                                                             

EXAMPLE 5 Fermentation Experiments

Rumen bacterial suspensions were prepared using rumen fluid obtainedfrom a cannulated steer. The final bacterial suspensions comprised agrowth medium which included 65 mL of centrifuged rumen fluid plus 65 mLof deionized water. Various levels of the triamide (of Example 3) from 1mg/mL solutions or suspensions in methanol were added to 50-mL vials.The methanol was evaporated, an 0.4 g of chopped, dried alfalfa (sieved,1 mm mesh) was added to each vial. The final bacterial suspensionsobtained as described above were added to the vials, 20 mL of suspensionper vial, and the suspensions were incubated for 20 hours with anaerobic CO₂ atmosphere, shaking at 39° C.

The suspensions were then processed for determination of volatile fattyacids (VFA). The VFAs were determined by the following procedure. Afterfiltration of the VFA sample through 0.2 micron filters to removeparticulate matter, samples were transferred to 2-mL vials. The VFAconcentrations of the samples were determined by GLC, using a HewlettPackard (HP) 5890A gas chromatograph, equipped with an HP 3392AIntegrator and an HP 7672A Automatic sampler. The column was a530-micron Carbowax 20M megabore capillary column, HP No. 19095-121. Thechromatograph was operated isothermally at 105° C., with the detectorand injection port set at 200° C., a flow rate of 17 cc/minute (heliumcarrier gas), an attenuation of 6, threshold setting of 5, peak width of0.01, area reject of 10,000, and a range setting of 0. Total run timewas 8 minutes. After about 300 samples had been run on the column, somepeak resolution was lost. To correct for the loss of resolution, thecarrier gas flow was reset to 14 cc/minute and the oven temperature wasreset to 100° C. Resolution and peak areas from standard solutionsindicated that no adjustment of peak area was necessary due to thechanges. The results of the VFA determinations are presented in Table 2.

The results indicated that the triamide compound was responsible for anincrease in the level of propionate in the rumen bacterial suspension,and this increase was greater with a larger dosage of the triamide. Inaddition, there were slight, but statistically significant decreases inacetate and butyrate levels.

                  TABLE 2                                                         ______________________________________                                        VFA CONCENTRATIONS IN                                                         TREATED BACTERIAL SUSPENSIONS                                                 Concentrations of VFAs (millimoles per liter)                                 SD = Standard Deviation                                                       Micro-                                                                        grams                                                                         of                                         To-                                Triamide                                                                             Ace-          Prop-                 tal                                per mL tic    SD     ionic SD   Butyric                                                                             SD   VFA  SD                            ______________________________________                                         0     50.1   0.9    19.7  0.3  5.1   0.1  74.9 1.3                           10     52.3   1.0    21.3  0.4  5.2   0.2  78.8 1.5                           20     48.1   1.3    20.2  0.7  5.1   0.2  73.4 2.2                           40     46.4   1.7    22.1  0.9  4.4   0.2  72.9 2.8                           80     43.0   3.4    24.8  2.0  3.7   0.4  71.5 5.8                           160    33.1   4.1    26.0  2.6  1.9   0.3  61.0 7.0                           ______________________________________                                    

EXAMPLE 6

Adaptation Experiments

The objective was to determine the long-term effects of the triamidecompound on fermentation by bacterial suspensions originally obtainedfrom the rumen of a rumen-cannulated steer. A growth medium was preparedusing 65 mL of centrifuged rumen fluid plus 65 mL of distilled water.The triamide (of Example 3) was added as 1 mg/mL solutions or supensionsin methanol to 20-mL vials for a nominal concentration of 50 mg/mL.

The methanol was evaporated and 0.2 g of chopped dried alfalfa (sieved,1 mm mesh) was added to each vial. Rumen-simulating medium was added toeach vial, and the vials were frozen until needed. Enough vials wereprepared each time for one week of the adaptation experiment.

On the first day, rumen contents were obtained from the rumen of arumen-cannulated steer and blended. The blended rumen contents wereadded to vials containing the experimental treatments, 3.0 mL per vial(final volume was 15 mL). All treatments were performed in duplicate.The vials were sealed, with an anaerobic CO₂ atmosphere, and incubated(shaking) at 39° C. After incubation for 24 hours, 3-mL volumes of thesuspensions were removed and transferred, by syringe and needle, toprewarmed (39° C.) vials containing the same medium and the sameexperimental chemical, as were in the original vials. The incubation wasrepeated, so that the each day's vials provided the inocula for the nextday of adaptation. The daily transfers were continued for six weeks.

Every day for the first week, and on alternating days of the remainingfive weeks, the volume of gas that was produced in the vials wasmeasured as follows: 100-mL serum vials were filled with water. Stopperswere fitted tightly into the tops of the vials, and needles wereinserted through the stoppers. Tubing was connected to one of theneedles, so that water could pass from the vial into a graduatedcylinder. Another section of tubing, with a needle at the other end, wasconnected to the second needle. To measure gas production, the secondneedle was inserted through the stopper of the fermentation vial. Thegas phase in the fermentation vial, which was under pressure because ofthe gas that was produced during the fermentation, passed through thetubing, and forced water out of the water-filled vial, and into thegraduated cylinder. The volume of water that passed into the graduatedcylinder was then measured.

A gas chromatographic (GC) method was employed to quantitate methane andhydrogen accumulation. Vials were stored in ice to prevent additionalgas production while the gases in other samples were being measured. A1-inch, 23-gauge needle, attached to a 1-cc tuberculin syringe, wasflushed with the gas phase from the fermentation vials, and was thenused to remove samples of gas from the vials. Samples (0.5 cc) of thegas were then injected into the GC. The GC was a Varian Model 3700,equipped with a Hewlett-Packard 5890A integrator. The oven was operatedat 145° C. Supelco I-2579 100/120 Carbosieve II molecular sieve, packedin a stainless-steel 1/8 inch OD, 10-foot-long column, was employed forseparation of gases. Helium was the carrier gas, flowing at a rate of 30cc/minute. A thermal conductivity detector was used so that all gasescould be measured, at a temperature of 100° C., with a filamenttemperature of 140° C. The injector temperature was 160° C. The signalanalysis settings were changed for the individual gases, because of thewide differences in gas concentrations and detector sensitivity to thegases. The attenuations were set for the individual gases as follows:hydrogen, -5; nitrogen/oxygen (co-elution), 2; methane, -2; carbondioxide, 2. Threshold settings were as follows: hydrogen, -1; all othergases, 0. Peak width settings were as follows: hydrogen, 0.04; allremaining gases, 0.16. Hydrogen eluted at 0.85 minutes, nitrogen/oxygeneluted at 1.91 minutes, methane eluted at 4.75 minutes, and carbondioxide eluted at 8.29 minutes. A standard was employed containing 1%hydrogen, 1% oxygen, 1% methane, and 1% carbon dioxide, in nitrogen.Because small amounts of air mixed into the syringe during the samplingprocedure, the amount of nitrogen/oxygen present in the GC run was usedto correct for leakage (For example, if the analysis indicated thepresence of 10% air, the values for methane and hydrogen were divided by0.9). Moles of gases produced were calculated using the percentages ofeach gas (by volume), the total volume of gas, and Boyle's gas law.Because the bulk gas was CO₂, the measurement of CO₂ using the GC wasnot accurate, but the measurement of total gas volume, minus theproportions of hydrogen and methane, provided an estimate of CO₂production.

During the first week, the gas volumes and the proportions of individualgases were measured on the same days. During subsequent weeks, the twomeasurements were performed on opposite days, in order to speed up theprocedure and prevent unnecessary contamination of the gas phase withair. For the computed values for individual gases, the gas volumesproduced on one day were used for calculation of the molar amounts ofthe gases, based on the GC data for the next day. Weekly averages forall values were calculated for this report. The suspensions were thenprocessed for determination of volatile fatty acids (VFA), and fordetermination of lactic acid, and the acids were measured by GC, asdescribed earlier. The results are presented in Tables 3 and 4.

The results again indicated that the triamide compound caused anincrease in the production of propionate as well as a decrease in theamount of methane produced. There was also a slight decrease in theamount of butyrate produced, and no significant change in the level ofacetate produced. Since propionate is a more efficient source of energyfor the ruminant animal than acetate or butyrate, greater feedefficiency will be the result of adding the triamide of the presentinvention to the feed of a ruminant animal. The observed reduction inthe amount of methane produced is also a good indication of increasedefficiency. Additionally, because the reduction in the production ofacetate will only be slight (it may or may not be measurable), thereshould be no significant change in the composition (fat content) of themilk produced when the triamide is fed to a lactating ruminant in orderto improve milk production.

                                      TABLE 3                                     __________________________________________________________________________    EFFECT OF TRIAMIDE ON FERMENTATION GAS PRODUCTION                                                            Volume                                               Micro-  Micro-           of                                                   Moles   Moles   Micro-   Gas cc                                         Week of                                                                             of      of      Moles of Per                                            Adaptation                                                                          CO.sub.2                                                                          SD  Methane                                                                            SD Hydrogen                                                                            SD Vial SD                                        __________________________________________________________________________    1. Control Suspension                                                         1     901 03   76  45 0     0  28.6 2.0                                       2     880 65  203  23 t     0  31.1 1.2                                       3     903 28  159  11 t     0  30.6 0.8                                       4     898 74  144  38 0     0  30.2 2.6                                       5     779 23   97   4 t     0  26.1 0.6                                       6     708 13   90   2 t     0  24.3 0.4                                       __________________________________________________________________________    2. Suspension With Triamide                                                   1     888 106  56  31 0     0  27.8 2.4                                       2     887 40  165  21 t     0  30.4 0.5                                       3     958  9  140  15 t     0  31.5 0.6                                       4     933 38  137  17 0     0  30.8 1.2                                       5     878 -36 105   7 t     0  28.8 1.0                                       6     803  9   98   5 t     0  26.8 0.4                                       __________________________________________________________________________     Values are the amounts of the gases produced per vial of suspension per       day, as weekly means (duplicate treatments, 2 to 5 determinations per         week). SD = standard deviation; t = trace (too small to measure)         

                  TABLE 4                                                         ______________________________________                                        EFFECT OF TRIAMIDE ON VFAs                                                    Week                                                                          of    Acetic           Propionic    Butyric                                   Adap- Acid             Acid         Acid                                      tation                                                                              Millimolar                                                                              SD     Millimolar                                                                            SD   Millimolar                                                                            SD                                ______________________________________                                        1. Control Suspension                                                         1     34.2      6.4     9.5    3.2  4.3     1.1                               2     32.0      5.5    10.7    2.7  4.8     1.2                               3     36.7      3.3    11.0    2.3  4.9     1.3                               4     40.5      2.7    13.2    1.7  6.5     0.5                               5     41.4      1.5    12.7    0.5  6.5     0.3                               6     27.9      1.7     6.7    0.5  3.0     0.2                               ______________________________________                                        2. Suspension With Triamide                                                   1     30.6      4.8     8.9    3.3  3.1     1.0                               2     35.7      8.5    12.6    5.2  3.6     1.6                               3     41.6      6.2    17.4    2.8  4.5     0.7                               4     39.9      4.2    15.4    2.9  4.9     0.8                               5     45.3      3.5    18.2    1.6  6.1     0.6                               6     24.0      8.3     7.7    1.1  2.4     0.3                               ______________________________________                                    

Values are the means for each week (duplicate samples, with two to fivemeasurements taken per week). Concentrations are given as millimolaramounts present in the fermentation mixture.

What is claimed is:
 1. A method for increasing the efficiency of feedutilization by ruminant animals comprising administering to said animalsan effective amount of one or more compounds having the formula:##STR9## wherein each R may be the same or different and represents astraight or branched chain alkyl group of one to six carbon atoms,or thephysiologically acceptable salts thereof.
 2. A method according to claim1 wherein the compound is administered orally.
 3. A method according toclaim 1 wherein said effective amount is from about 0.2 to about 100mg/kg of body weight of the animal treated per day.
 4. A methodaccording to claim 1 wherein said effective amount is from about 1 toabout 10 mg/kg of body weight of the animal treated per day.
 5. A methodaccording to claim 1 wherein R contains three to five carbon atoms.
 6. Amethod according to claim 1 wherein R is selected from the groupconsisting of tert-butyl, n-butyl, isobutyl, and isopropyl.
 7. A methodaccording to claim 1 wherein R is isobutyl.